to locomotion ( 3 , 19 – 21 ), but other brain areas
have been proposed to contribute to this os-
cillation. One such area is the supramammil-
lary nucleus (SuM) of the posterior hypothalamus
( 22 – 24 ), which has recently been shown to
have roles in arousal ( 25 ), spike-timing coor-
dination ( 26 ), and identification of novelty ( 27 ).
As a proposed theta controller, neural activity
in the SuM is likely also related to locomotion,
but this has not been systematically inves-
tigated. In addition to innervating the medial
septum, the SuM has highly divergent outputs
that target the midbrain, where locomotor
commands are integrated, as well as regions
involved in spatial navigation, including the
hippocampus, entorhinal cortex, medial pre-
frontal cortex, nucleus reuniens, and claustrum
( 28 ). Thus, the SuM projects to theta, locomo-
tor, and spatial navigation circuitry, but the
functional relevance of this positioning remains
poorly understood.
Using electrophysiological data from rats
navigating a continuous alternation task for
reward ( 26 ), we first investigated how the
spiking activity of SuM neurons relates to
locomotor speed and hippocampal theta oscil-
lations (Fig. 1A). Similar to previous observa-
tions in the midbrain locomotor region (MLR)
( 29 ), we found a large proportion of SuM units
with firing rates that were significantly coupled
to locomotor speed, with the majority display-
ing a positive correlation (Fig. 1, B and C, and
fig.S1,A,C,andD).Notably,SuM“speed cells”
were more closely correlated with future speed
(with an average offset of 1.2 s) than real-time
speed (Fig. 1D and fig. S1, A and D). After
adjusting for temporal offsets, the firing rates
of 99% of SuM units were modulated by speed
(fig. S1B). Most speed cells retained their cor-
relation with immobility data withheld (fig.
S1, E and F) and were more weakly coupled
to acceleration than speed (fig. S2). SuM unit
activity was also correlated to hippocampal
theta amplitude in real time, but this was
less accurately modeled than speed (fig. S3).
Thus, SuM activity and locomotion are strongly
coupled.
We then addressed whether speed-related
neuronal activity is propagated to projection
targets, because not all locally recorded SuM
units are necessarily projection neurons.
In vivo two-photon calcium imaging of SuMaxon terminals that innervate the dentate
gyrus (DG) and the CA2 region of the hippo-
campus was performed on head-fixed mice
(fig. S4A). Indeed, speed-correlated activity
in SuM axons was observed in both regions
(fig. S4, B and C). Extensive collateralization
of SuM axons was also determined (fig. S5)
and may contribute to the similar proportion
of positively and negatively correlated axons
in both regions (fig. S4B).
Next, we examined the coupling between
SuM action potentials and hippocampal theta
waves (Fig. 1E). High coherence with hippo-
campal theta oscillations and theta-rhythmic
spiking was observed for 30.7% of units (Fig. 1,
F and G; see materials and methods). SuM
theta cells typically fired near the trough of
CA1 theta waves, with a slight prospective bias
(fig.S6).MuchliketheoverallSuMpopulation,
most SuM theta cells were positively correlated
with locomotor speed (Fig. 1H).
To test the functional involvement of the
SuM in locomotion and theta activity, we
injected the SuM with recombinant adeno-
associated virus (rAAV) to express the opto-
genetic proteins channelrhodopsin-2 (ChR2;SCIENCEscience.org 17 DECEMBER 2021•VOL 374 ISSUE 6574 1493
Fig. 2. Optogenetic SuM modulation controls locomotion and hippocampal
LFP.(A) Pan-neuronal SuM activation with ChR2 (blue) or inhibition with HR
(orange) in head-fixed mice on a floating ball. (B) Bidirectional locomotor effect with
SuM activation (top) and inhibition (bottom). Colored bars denote“laser on”
segments. (C) Percent of trials where locomotion was initiated or halted for ChR2
(top) and HR (bottom). (D) Latency of the start versus stop response. (E) Speed
during locomotor epochs before (Pre) and during (On) light delivery. ChR2,t 5 =−1.14,
P= 0.31; HR,t 4 = 3.07,P= 0.037. (F) Percent of time spent locomoting before
(Pre) and during (On) light delivery. ChR2,t 5 =−4.84,P= 0.0047; HR,t 4 = 4.60,P=
0.010. (G)(Top)Optogeneticactivationat4,8,or12Hzcomparedwithnolaser
control. Blue bars denote laser on. (Bottom) Optogenetic inhibition (orange shading)
versus no laser control. The scale bar applies across rows. (H) Quantification of
power spectrum changes normalized to no laser control. (Top) ChR2: 4-Hz power at 4-Hz
stimulation,t 5 = 0.86,P= 0.43; 8-Hz power at 8-Hz stimulation,t 5 = 5.18,P= 0.0035;
12-Hz power at 12-Hz stimulation,t 5 = 3.99,P= 0.010. (Bottom) HR:t 4 = 0.043,
P= 0.97. Data are mean ± SEM. ns, not significant. *P< 0.05, **P< 0.01.RESEARCH | REPORTS